Several publications and patent documents are cited throughout the specification in order to describe the state of the art to which this invention pertains. Each of these citations is incorporated herein by reference as though set forth in full.
Wild-type AAV is a parvovirus with a ˜4.7 kb single-stranded DNA genome. The virus is naturally replication-defective and requires a helper virus such as adenovirus or herpesvirus for replication. The virus has not been associated with any disease but instead was initially isolated as a contaminant of adenoviral isolates (4). Six serotypes have been described, with highly conserved sequences (varying from 62-99%). The viral genome is flanked by two inverted terminal repeats (ITRs), and encodes three capsid genes (VP1, 2, 3) and 4 rep proteins involved in DNA replication and in control of the AAV life cycle. Three additional serotypes (AAV-7, -8, -9) have recently been isolated from Rhesus macaques and humans and are also >60% conserved compared to AAV-1-6 (5).
Wild-type AAV has been engineered for use as a gene delivery vehicle. The rep and cap genes are deleted, and the therapeutic gene of interest inserted between the two ITRs, such that there is no coding viral DNA. In the mid-1990's several groups (6-10) showed that recombinant AAV could infect multiple non-dividing cell types, including skeletal muscle, liver, CNS, and respiratory tract, and could direct long-term expression of a transgene in an immunologically competent animal. This exciting finding has been exploited by a number of groups and there is now an impressive portfolio of results in which genetic diseases have been cured in small and large animal models by the administration of recombinant AAV (11-17). Experience in humans is more limited (18-24), but has been promising in terms of safety and of evidence for gene transfer and expression, although levels of expression have not yet been high enough to produce phenotypic correction in most instances.
One major objective of our research is the establishment of a safe and effective adeno-associated virus (AAV)-mediated gene transfer system for treating hemophilia and other blood coagulation disorders. Based on long-term cure of hemophilia in the canine model of the disease (1), a clinical study was designed in which subjects with severe hemophilia B were infused via the hepatic artery with AAV-F.IX. One subject achieved circulating Factor IX levels of 11.8% (therapeutic range) by the second week after vector infusion. These levels were sustained for approximately four weeks and then gradually began to fall, eventually returning to the subject's baseline level of <1%. Coincident with the fall in F.IX levels, the liver transaminase enzymes in the blood began to rise, peaking at 5 weeks after infusion, and declining to normal several weeks thereafter. Thus, the subject pursued a course quite different from that seen in experimental animals, including mice, rats, rabbits, hemophilic dogs, and non-human primates. In contrast to experimental animals, the human subject had pre-existing immunity to AAV-2, as evidenced by the presence of a low neutralizing antibody titer to AAV; and by inference from the presence of IgG antibodies, the subject also likely had a population of AAV-specific memory T cells in his lymphoid compartment (2). Similar findings were observed in another subject in the trial, and immunologic studies in this subject documented a T cell response to a specific peptide in the AAV capsid. Notably, the response was detectable in the peripheral blood for several weeks after, but not before vector infusion.
In light of these findings, it is clear that in order for gene therapy approaches to be effective, in certain instances, it may be necessary to modulate the immune response to prevent T-cell mediated destruction of transgene expressing cells.